Use of Thermostable RNA Polymerases to Produce RNAs Having Reduced Immunogenicity

ABSTRACT

Provided herein, among other things, is a method for producing an RNA product that has reduced immunogenicity. In some embodiments, the method involves transcribing a template DNA with a thermostable RNA polymerase at a temperature of greater than 44° C.

BACKGROUND

Synthetic RNAs are a promising new class of therapeutics fornon-virus-mediated gene therapy, vaccines and protein replacementtherapeutics, as well as in immuno-oncology and personalized cancervaccines (Sahin, et al., (2014): Nature Reviews Drug Discovery 13,759-80; Weissman, (2015), Expert Review of Vaccines, 14(2):265-81).Synthetic RNAs are commonly manufactured by in vitro transcription (IVT)of a DNA template that encodes the antigen or protein of interest(Sahin, et al., (2014); Steinle, et al., (2017), Stem Cells,35(1):68-79).

One limitation associated with the therapeutic use of synthetic RNA isan immunostimulatory response induced by double-stranded RNA (dsRNA)contaminants created during IVT (Devoldere, et al., (2016), DrugDiscover Today, 21(1), 11-25; Loomis et al., (2016), Journal ofMaterials Chemistry 4:1619-32; Triana-Alonso et al., (1995), The Journalof Biological Chemistry, 270 (11): 6298-6307). The immunostimulatoryresponse of cells results from activation of receptors that triggersecretion of interferons and inflammatory cytokines (Devoldere et al.,(2016); Loomis et al., (2016); Kariko, et al., (2005) Immunity,23:165-175).

Several methods have been developed to separate desired IVT productsfrom contaminating dsRNA. These include various chromatographytechniques such as: ion exchange high-performance liquid chromatography(HPLC), reverse phase HPLC, hydrophobic interaction HPLC, low or normalpressure liquid chromatography, size exclusion chromatography, oligo dTaffinity chromatography, and core bead chromatography (Kariko, et al.,(2011) Nucleic Acids Research, 39 (21), e142; Weissman, et al., (2012)Methods in Molecular Biology, 969:43-54; Kobuk, et al., (2013) RNA,10:1449-59; US 2016/0024141; US 2016/0024140 A1; U.S. Pat. No.8,383,340; WO 2014/144711; US 2016/0032316; US 2014/144767; US2016/0326575). Enzymatic digestion of dsRNA with RNase III, RNase V1,Dicer, and Chipper is also implemented to reduce dsRNA (US2016/0032316).

The use of a physical separation method to remove the dsRNA from IVTreactions increases the cost and labor involved in the production of IVTRNAs that minimally activate innate immune responses.

SUMMARY

It has been found that IVT of a DNA template at an elevated temperature(e.g., at a temperature of greater than 44° C.) produces an RNA productthat is less immunostimulatory than RNA products that are produced at alower temperature (at 37° C.). Without wishing to be bound to anyspecific theory, it is believed that RNA products produced at anelevated temperature are less immunostimulatory than those produced at alower temperature because they contain less dsRNA, which is known tohave an immunostimulatory effect. As such, in some embodiments, the RNAproducts produced at an elevated temperature can be transfected intocells without first removing the dsRNA from the RNA product, i.e.,without first purifying the non-dsRNA products from the RNA product(such as using chromatography or degrading the dsRNA enzymatically).

A variety of methods and compositions are described herein. In someembodiments, the method may comprise: (a) transcribing a template DNAwith a thermostable RNA polymerase at a temperature of greater than 44°C. to produce an RNA product; and (b) introducing (e.g. transfecting)the RNA product into mammalian cells. Because the RNA product producedby this method does not contain significant amounts of dsRNA (has areduced dsRNA content as compared to a control RNA product produced fromthe same template using the same RNA polymerase but at a lowertemperature of only 37° C.), the method may be done in the absence of astep that removes dsRNA from the RNA product prior to introducing theRNA product into the mammalian cells.

Embodiments provide a method comprising: (a) transcribing a template DNAwith a thermostable RNA polymerase at a temperature of greater than 44°C. (such as at a temperature of at least 50° C.) to produce an RNAproduct; and (b) measuring the immunogenicity of the RNA product in theabsence of a step that removes any dsRNA from the RNA product.

The thermostable RNA polymerase may be a variant of a bacteriophage RNApolymerase, such as a thermostable variant of the wild type T7 RNApolymerase having the amino acid sequence shown in SEQ ID NO:1.

The RNA product may be a protein, such as a therapeutic protein. The RNAproduct may be a therapeutic RNA and/or may be a guide RNA, a shorthairpin RNA, a siRNA, a microRNA, a long noncoding RNA, an mRNA encodinga recombinant protein or a native protein, an RNA containing modifiednucleotides, and a capped mRNA.

The immunogenicity of the RNA product may be measured by any knownmeans. For example, the immunogenicity may be measured by assaying theRNA product for the presence of dsRNA. As discussed herein, dsRNA isknown to have an immunostimulatory effect. The presence or amount ofdsRNA can therefore be correlated with immunogenicity of the RNAproduct. Alternatively, or in addition, the immunogenicity of the RNAproduct may be measured by introducing the RNA product into one or moremammalian cells. The mammalian cells may be cells of a mammalian subjectin vivo (i.e. the RNA product is administered to a mammal, such as atest mammal; and an immune response in the mammal is measured).Alternatively, or in addition, the mammalian cells may be mammaliancells cultured in vitro or mammalian cells ex vivo (in which case theRNA product is introduced into the cells, such as by transfection; andan immune response in the cells is measured, such as using anenzyme-linked immunosorbent assay (ELISA) to determine the level of e.g.IFN-α and/or TNF-α in the cell supernatant).

The method may involve comparing the immunogenicity measured for the RNAproduct with the immunogenicity measured for a control RNA product. Thecontrol RNA product may be produced by transcribing the template DNAwith the thermostable RNA polymerase at a temperature of 37° C., in theabsence of a step that removes any dsRNA from the control RNA. Thus, themethod may further comprise: (i) producing a control RNA product bytranscribing the template DNA with the thermostable RNA polymerase at atemperature of 37° C. and, in the absence of a step that removes anydsRNA from the control RNA product, measuring the immunogenicity of thecontrol RNA product; and (ii) comparing the immunogenicity measured forthe RNA product with the immunogenicity measured for the control RNAproduct. The immunogenicity of the control RNA product may be measuredby any known means; such as by assaying the control RNA product for thepresence of dsRNA, by introducing the control RNA product into cells ofa mammalian subject in vivo, or by introducing the control RNA productinto mammalian cells cultured in vitro or into mammalian cells ex vivo.

Embodiments also provide a method comprising: (a) transcribing atemplate DNA with a thermostable RNA polymerase at a temperature ofgreater than 44° C. to produce an RNA product; and optionally measuringthe immunogenicity of the RNA product (e.g. using any techniquedescribed herein); and (b) combining the RNA product with apharmaceutically acceptable excipient; wherein the method is done in theabsence of a step that removes any dsRNA from the RNA product betweensteps (a) and (b).

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIGS. 1A-1B shows the presence of dsRNA contaminants in IVT RNAs fromdifferent DNA templates that were synthesized under standard conditions(37° C.). The DNA templates used were pXba SalI (6 kb), pXba HpaI (9kb), pXba AvrII (2.5 Kb), and Cluc NotI (3 kb). Crude IVT reactions weresubjected to immunoblot analyses using a monoclonal antibody (mAb-J2)specific for any form of dsRNA (Schonborn, et al., (1991), Nucleic AcidsResearch 19(11) 2993-3000; obtained from English and ScientificConsulting, Budapest, Hungary). The intensity of the blackness on theimmunoblot correlates with antibody binding to dsRNA contaminants.

FIG. 1A shows the results from transcription with wild-type T7 RNApolymerase at 37° C.

FIG. 1B shows the results from transcription with a thermostable variantof T7 RNA polymerase at 37° C. At 37° C., both wild-type and thethermostable variant of RNA polymerase generated the desired RNA product(1.2% agarose gel) and contaminating dsRNA (immunoblot) regardless ofthe length of the DNA template.

FIGS. 2A-2D shows the reduction in amounts of dsRNA in IVT reactionsthat were performed at higher temperatures either because the wild-typeT7 polymerase was inactive or because the thermostable T7 polymerase didnot produce dsRNA. Activity was determined by the amount of IVT RNAobserved on the 1.2% agarose gel. The observed effect was independent ofthe length of the DNA template.

FIG. 2A shows, using wild-type T7 RNA polymerase, dsRNA detected by mAbJ2 (English and Scientific Consulting, Budapest, Hungary) in IVTreaction mixtures at temperatures between 37° C. and 55° C. Thepolymerase activity is lost at temperatures greater than 43.9° C., asdetermined by the absence of IVT Cluc NotI RNA. Moreover, dsRNA wasdetected in IVT reaction mixtures using the mAb J2 (English andScientific Consulting, Budapest, Hungary) at temperatures between 37° C.and 43.9° C.

FIG. 2B shows, using a thermostable T7 polymerase, dsRNA detected by mAbJ2 in IVT reaction mixtures at temperatures between 37° C. and 55° C.The detectable amount of dsRNA is substantially reduced at temperaturesgreater than 44° C. while the amount of IVT Cluc NotI RNA produced atthe same time using thermostable T7 RNA polymerase is significant.

FIG. 2C shows, using wild-type T7 RNA polymerase, that both the amountof IVT RNA and dsRNA from DNA templates of different lengths aresubstantially reduced at temperatures of 55° C.

FIG. 2D shows, using a thermostable T7 RNA polymerase, that only dsRNAcontaminants in IVT mix from DNA templates of different lengths aresubstantially reduced at temperatures of 55° C. while at the same timethe yields of IVT were significant and similar throughout.

FIG. 3 shows the effect of high temperature on dsRNA formation in IVTreactions using a commercially available thermostable T7 RNA polymerasefrom Toyobo Life Science Department, Osaka, Japan on Cluc NotI templateDNA. IVT was performed at 37° C. or 50° C. Only the amount of dsRNAcontaminants was reduced at 50° C. while significant amounts of ClucNotI RNA were detected on a 1.2% agarose gel. This data demonstratesthat the temperature of the reaction rather than the particularthermostable T7 RNA polymerase is responsible for reduction of dsRNA.

FIG. 4 shows the temperature dependent reduction of dsRNA associatedwith IVT of Cluc NotI RNA that either lacks a poly A tail (no tailing)or was polyadenylated with a tail-length of 125 nucleotides (T125). BothRNAs contained a modified nucleotide-pseudouridine instead of uridine.

FIGS. 5A-5B shows activation of interferons and cytokines (representedby IFN-α and TNF-α respectively) indicative of an immune responseactivation in human dendritic cells (hDCs) that were transfected withCluc NotI IVT RNA from reactions that were performed with wild-type T7RNA polymerase at 37° C. or with a thermostable variant of T7 RNApolymerase at 55° C. Poly I:C, a synthetic analog of dsRNA andResiquimod (R848), an activator of Toll-like receptors are used ascontrols for interferon activation. Negative controls included TransIT®(Mirus Bio, Madison, Wis.) transfection reagent alone and PBS.

FIG. 5A shows the results of absolute quantification of IFN-α(interferon) levels in the cell culture supernatants of hDCs that weretransfected with Cluc NotI IVT RNA using ELISA (Kariko, et al., (2011)).Cluc NotI IVT RNA (or control RNA-poly I:C) was introduced into hDCs,and supernatants were collected 24 hours after transfection. Thesupernatants were then probed for the secretion of IFN-α. Higherinterferon secretion is observed with Cluc NotI IVT RNA from 37° C.transcription reactions without subsequent removal of the dsRNA (IVT 37°C.) as compared to Cluc NotI IVT RNA from 55° C. transcription reactionswithout subsequent removal of the dsRNA (IVT 55° C.) or HPLC-purifiedCluc NotI IVT RNA (IVT 37° C._HPLC) indicating low immunostimulatoryproperties of IVT RNA synthesized at 55° C. Increased secretion of IFN-αis seen with polyl:C (positive control). Total rat RNA, that is known tohave reduced immunogenicity, was also used as a control.

FIG. 5B shows the absolute quantification of Tumor necrosis factor(TNF)-α (cytokine) levels in supernatants of hDCs that were transfectedwith Cluc NotI IVT RNA using ELISA (Kariko, et al., (2011)). Cluc NotIIVT RNA (or control RNA-poly I:C) were introduced into hDCs, andsupernatants were collected 24 hours after transfection. Thesupernatants were then probed for the secretion of TNF-α. Highercytokine secretion is observed with Cluc NotI IVT RNA from 37° C.transcription reactions without subsequent removal of the dsRNA (IVT 37°C.) as compared to Cluc NotI IVT RNA from 55° C. transcription reactionswithout subsequent removal of the dsRNA (IVT 55° C.) or HPLC-purifiedCluc NotI IVT RNA (IVT 37° C._HPLC) indicating low immunostimulatoryproperties of IVT RNA synthesized at 55° C. Resquimod (R848), animidazoquinoline compound, used as a positive control showed increasedcytokine secretion. Total rat RNA, that is known to have reducedimmunogenicity, was used as a control.

DETAILED DESCRIPTION

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are described.

All patents and publications, including all sequences disclosed withinsuch patents and publications referred to herein, as well as U.S.Provisional Application Ser. No. 62/522,877 filed Jun. 21, 2017, andU.S. patent application Ser. No. 15/820,656 filed Nov. 22, 2017, areexpressly incorporated by reference.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention. Accordingly, the terms definedimmediately below are more fully defined by reference to thespecification as a whole.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

As used herein, the term “in vitro transcription” (IVT) refers to acell-free reaction in which a DNA template (e.g. a double-stranded DNAtemplate) is copied by a DNA-directed RNA polymerase to produce aproduct that contains RNA molecules that have been copied from thetemplate.

As used herein, the term “DNA template” or “template DNA” refers to adouble-stranded DNA molecule that is transcribed in an IVT reaction. DNAtemplates have a promoter (e.g., a T7, T3 or SP6 promoter) recognized bythe RNA polymerase upstream of the region that is transcribed.

As used herein, the term “RNA product” refers to the product of an IVTreaction. The RNA product of IVT contains a mixture of RNA moleculesand, depending on how the transcription is done, may contain dsRNAmolecules. The molecular events that generate dsRNA molecules in IVTreactions is unknown. dsRNA molecules can be detected using an antibodythat is specific for dsRNA or by liquid chromatography (e.g., HPLC), forexample.

As used herein, the terms “less immunostimulatory” and “lessimmunogenic” (or “reduced immunostimulation” or “reducedimmunogenicity”) are used interchangeably to describe a reduction in animmune response (e.g., a reduction of interferon or cytokine expression)relative to a reference sample, e.g., a control. A decrease inimmunostimulation or immunogenicity may be a response that is reduced byat least 20%, at least 40%, at least 60%, at least 80%, at least a 90%,or at least a 95% relative to the control.

As used herein, the terms “reduced dsRNA”, “less dsRNA” and “fewer dsRNAmolecules” are used interchangeably to refer to a sample that has anamount of dsRNA that is at least 20%, at least 40%, at least 60%, atleast 80%, at least 90%, or at least 95% less than the amount of dsRNAin a reference or control sample.

As used herein, the term “thermostable RNA polymerase” refers to an RNApolymerase that has a temperature optimum of greater than 44° C., suchas at a temperature optimum of at least 50° C., at least 55° C., or atleast 60° C. In many embodiments a thermostable RNA polymerase may be avariant of a wild type mesophilic RNA polymerase, where the wild typemesophilic RNA polymerase is substantially inactive at the temperatureat which the thermostable variant is optimally active. The thermostableRNA polymerase may be purified before use. The RNA polymerase may bestored in a storage buffer before being added to a reaction mixture ortherapeutic formulation.

As used herein, the term “step that removes dsRNA” refers to any methodthat can be used to specifically remove dsRNA, but not RNA that is notdsRNA, from a sample. For example, dsRNA can be removed bychromatography (e.g., HPLC). In another example, dsRNA can be removedusing an RNase that is specific for dsRNA, e.g., RNase III, RNase V1,Dicer, or Chipper. A step that removes dsRNA from a sample does not needto remove all of the dsRNA from the sample. Rather, such a step shouldremove at least 80%, at least 90% or at least 95% (up to 100%) of thedsRNA from the sample. A method that is performed “in the absence of astep that removes dsRNA” does not include any method step thatspecifically removes dsRNA, but not RNA that is not dsRNA, from a sample(such as but not limited to the dsRNA-removing steps discussed above).

As used herein, the term “variant” refers to a protein that comprises orconsists of an amino acid sequence that is different from a reference(e.g. naturally occurring) amino acid sequence (i.e., having less than100% sequence identity to the amino acid sequence of a reference (e.g.naturally occurring protein)) but that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 98% or at least 99%identical to the reference (e.g. naturally occurring) amino acidsequence.

As used herein, the term “introducing” refers to any means forintroducing a nucleic acid into cell, including, but not limited to,transfection, microinjection, electroporation and lipid-mediatedmethods.

As used herein, the term “buffering agent”, refers to an agent thatallows a solution to resist changes in pH when acid or alkali is addedto the solution. Examples of suitable non-naturally occurring bufferingagents that may be used in the compositions, kits, and methods of theinvention include, for example, Tris, HEPES, TAPS, MOPS, tricine, orMES.

The term “non-naturally occurring” refers to a composition that does notexist in nature.

Any protein described herein may be non-naturally occurring, where theterm “non-naturally occurring” refers to a protein that has an aminoacid sequence and/or a post-translational modification pattern that isdifferent from the protein in its natural state. For example, anon-naturally occurring protein may have one or more amino acidsubstitutions, deletions or insertions at the N-terminus, the C-terminusand/or between the N- and C-termini of the protein. A “non-naturallyoccurring” protein may have an amino acid sequence that is differentfrom a naturally occurring amino acid sequence (i.e., having less than100% sequence identity to the amino acid sequence of a naturallyoccurring protein) but that is at least 80%, at least 85%, at least 90%,at least 95%, at least 97%, at least 98% or at least 99% identical tothe naturally occurring amino acid sequence. In certain cases, anon-naturally occurring protein may contain an N-terminal methionine ormay lack one or more post-translational modifications (e.g.,glycosylation, phosphorylation, etc.) if it is produced by a different(e.g., bacterial) cell. A “mutant” protein may have one or more aminoacid substitutions relative to a wild-type protein and may include a“fusion” protein. The term “fusion protein” refers to a protein composedof a plurality of polypeptide components that are unjoined in theirnative state. Fusion proteins may be a combination of two, three or evenfour or more different proteins. The term polypeptide includes fusionproteins, including, but not limited to, a fusion of two or moreheterologous amino acid sequences, a fusion of a polypeptide with: aheterologous targeting sequence, a linker, an epitope tag, a detectablefusion partner, such as a fluorescent protein, β-galactosidase,luciferase, etc., and the like. A fusion protein may have one or moreheterologous domains added to the N-terminus, C-terminus, and or themiddle portion of the protein. If two parts of a fusion protein are“heterologous”, they are not part of the same protein in its naturalstate.

In the context of a nucleic acid, the term “non-naturally occurring”refers to a nucleic acid that contains: a) a sequence of nucleotidesthat is different from a nucleic acid in its natural state (i.e., havingless than 100% sequence identity to a naturally occurring nucleic acidsequence), b) one or more non-naturally occurring nucleotide monomers(which may result in a non-natural backbone or sugar that is not G, A, Tor C) and/or c) may contain one or more other modifications (e.g., anadded label or other moiety) to the 5′-end, the 3′ end, and/or betweenthe 5′- and 3′-ends of the nucleic acid.

In the context of a preparation, the term “non-naturally occurring”refers to: a) a combination of components that are not combined bynature, e.g., because they are at different locations, in differentcells or different cell compartments; b) a combination of componentsthat have relative concentrations that are not found in nature; c) acombination that lacks something that is usually associated with one ofthe components in nature; d) a combination that is in a form that is notfound in nature, e.g., dried, freeze dried, crystalline, aqueous; and/ore) a combination that contains a component that is not found in nature.For example, a preparation may contain a “non-naturally occurring”buffering agent (e.g., Tris, HEPES, TAPS, MOPS, tricine or MES), adetergent, a dye, a reaction enhancer or inhibitor, an oxidizing agent,a reducing agent, a solvent or a preservative that is not found innature.

In some embodiments, a method for introducing an IVT RNA product intomammalian cells is provided. In embodiments, the IVT RNA being testeddoes not contain pseudouridine. The method may be performed in vitro orin vivo. For example, the mammalian cells may be in vitro or may be exvivo. Alternatively, the IVT RNA product may be introduced intomammalian cells by administering the IVT RNA product to a mammaliansubject. In any of these embodiments, the method may comprise: (a)transcribing a template DNA with a thermostable RNA polymerase at atemperature of greater than 44° C. (e.g., a temperature of at least 45°C., at least 50° C., at least 55° C. or at least 60° C., up to about 70°C. or 75° C.) to produce an RNA product; and (b) introducing the RNAproduct into mammalian cells. The RNA product is generally introducedinto mammalian cells at temperatures of about 37° C. Because the RNAproduct contains significantly reduced amounts of dsRNA as compared toan RNA product (made in the presence of U, A, G and C nucleotides)produced by transcription from the template DNA with the polymerase at alower temperature of e.g. about 37° C., the method may be done in theabsence of a step that removes any dsRNA from the RNA product (i.e., apurification step or enzyme treatment step) prior to introducing the RNAproduct into the cells, i.e., between steps (a) and (b) of the method.In this method, the RNA product obtained in step (a) and introduced intothe cells in step (b) is believed to be less immunostimulatory than acontrol RNA product produced by transcribing the template DNA with thepolymerase at a temperature of 37° C., when the immunostimulatory effectof the control RNA (containing nucleotides U, A, G and C but notmodified nucleotides of U) is evaluated by introducing the control RNAinto the cells in the absence of a step that would remove dsRNA fromcontrol RNA product. As illustrated below, the immunostimulatory effectof an RNA product can be measured by introducing the RNA product tomammalian cells and measuring the expression of markers for innateimmunity (e.g., interferons and cytokines, among many others) by thecells. Immunogenicity may be measured by ELISAs (e.g. as describedpreviously (Kariko, et al., (2011); Weissman, et al., (2012)). Forexample, mammalian cells are transfected with the IVT RNA product, thecells are harvested, and the cell supernatant is assayed for levels ofIFN-α and/or TNF-α.

Because the results shown below indicate that this effect appears to bebased on the temperature of the IVT reaction rather than the polymeraseused, the method may be done using any suitable thermostable variants ofa bacteriophage RNA polymerase. In some embodiments, the polymerase maybe a thermostable variant of the T7, T3 and SP6 RNA polymerases, whichhave been well characterized. Guidance for making thermostable variantsof those RNA polymerases can be found in PCT/US2017/013179 and U.S.application Ser. No. 15/594,090. In some embodiments, the thermostableRNA polymerase used in the method may be a variant of the wild type T7RNA polymerase of SEQ ID NO:1. In particular embodiments, thethermostable RNA polymerase may have an amino acid sequence that is atleast 80% or at least 90% identical to SEQ ID NO:1 (but less than 100%identical to SEQ ID NO:1) and may comprise an amino acid substitution atone or more positions corresponding to positions selected from 109, 205,388, 534, 567 and 618 of SEQ ID NO:1, as described in PCT/US2017/013179.For example, the variant may include a mutation at a positioncorresponding to 567 in SEQ ID NO:1 for example V567P.

The amino acid sequence of the full length T7 RNApolymerase is shown below (SEQ ID NO: 1)MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

In some embodiments, the RNA product may encode a protein, e.g., atherapeutic protein or a protein expected to alter the cells into whichit is introduced and, as such, the RNA molecules in the RNA product mayhave a 5′ untranslated region (5′ UTR), one or more coding sequences,and a 3′ translated region (3′ UTR), where the 3′ and 5′ UTRs facilitatetranslation of the one or more coding sequence to produce a proteinwithin the cells. In other embodiments, the RNA product may be atherapeutic RNA. In some embodiments the RNA product may be a guide RNA,a short hairpin RNA, a siRNA, a microRNA, a long noncoding RNA, or aprotein-coding RNA (which may encode a recombinant protein or a proteinthat is native to the cells). In some embodiments, the RNA product maycontain modified nucleotides (triphosphates for which can be added tothe IVT reaction). In these embodiments, modified nucleotides may beincorporated into the IVT RNA. Incorporation of modified nucleotides canincrease in translation efficiency of the RNA and increased stability ofthe RNA. Modifications can be present either in the sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/orin the phosphate groups (e.g., phosphorothioates and5′-N-phosphoramidite linkages); and/or in the nucleotide base (forexample, see: U.S. Pat. No. 8,383,340; WO 2013/151666; U.S. Pat. No.9,428,535 B2; US 2016/0032316). In some embodiments, the RNA product maybe altered during or after the transcription reaction, e.g., to decreasethe rate at which the RNA products are degraded in the cells. In someembodiment, the RNA product may contain capped RNAs (see, for example:WO 2016/090262; WO 2014/152673; WO 2009/149253; WO 2009/149253;Strenkowska, et al., (2016), Nucleic Acids Research, 44(20):9578-90).RNAs with poly A tails of varying length and labeled RNAs can also beproduced.

In some embodiments, the method may further comprise testing the RNAproduct for an immune-stimulatory effect, without performing a step thatremoves the dsRNA from the RNA product. As noted above, this may be donein a variety of different ways. The method may comprise measuring theimmunogenicity of the RNA product obtained in step (a) before or afterstep (b). In some embodiments, the method comprises the steps ofcomparing the immunogenicity of the RNA product with the immunogenicityof a control RNA product. For example, the method may comprise the stepsof: (i) producing a control RNA product by transcribing the template DNAwith the thermostable RNA polymerase at a temperature of 37° C. and, inthe absence of a step that removes any dsRNA from the control RNAproduct, measuring the measuring the immunogenicity of the control RNAproduct; and (ii) comparing the immunogenicity measured for the RNAproduct with the immunogenicity measured for the control RNA product.For example, in some embodiments, the amount of dsRNA in the RNA productmay be measured using a dsRNA-specific antibody or by liquidchromatography, for example.

In any embodiment, the in vitro transcription may be done using naturalNTPs, i.e., GTP, CTP, UTP and ATP to produce a product that does notcontain not contain modified nucleosides.

In any embodiment, the in vitro transcription may be done using NTPscorresponding to G, C, U and A in the absence of pseudo-uridinetriphosphate to produce a product that does not contain not containpseudo-uridine. The cells into which the RNA product is introduced maybe in vitro (i.e., cells that have been cultured in vitro on a syntheticmedium). In these embodiments, the RNA product may be transfected intothe cells. In other embodiments, the cells into which the RNA product isintroduced may be in vivo (cells that are part of a mammal). In theseembodiments, the introducing may be done by administering the RNAproduct to a subject in vivo. In some embodiments, the cells into whichthe RNA product is introduced may present ex vivo (cells that are partof a tissue, e.g., a soft tissue that has been removed from a mammal orisolated from the blood of a mammal).

Methods for making a formulation are also provided. In some embodiments,the method may comprise combining an RNA product made by transcribing atemplate DNA with a thermostable RNA polymerase at a temperature ofgreater than 44° C. with a pharmaceutically acceptable excipient toproduce a formulation. In some embodiments, the RNA product may becombined with the pharmaceutically acceptable excipient in the absenceof a step that removes any dsRNA from the RNA product. In someembodiments, the method comprises (a) transcribing a template DNA with athermostable RNA polymerase at a temperature of greater than 44° C. toproduce an RNA product; and (b) combining the RNA product with apharmaceutically acceptable excipient; wherein the method is done in theabsence of a step that removes any dsRNA from the RNA product betweensteps (a) and (b).

In some embodiments, the RNA can be formulated in a suitable excipientand effective therapeutic dose for introducing into a host for achievinga desired therapeutic effect. The formulation should lack adverseimmune-stimulatory effects caused by dsRNA.

The product RNA is not packaged into a virus particle prior toadministration.

In some embodiments, this method may further comprise administering theformulation to a subject, where the subject may be a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). Depending on the subject, the RNA (modified orunmodified) can be introduced into the cell directly by injecting theRNA or indirectly via the surrounding medium. Administration can beperformed by standardized methods. The RNA can either be naked orformulated in a suitable form for administration to a subject, e.g., ahuman. Formulations can include liquid formulations (solutions,suspensions, dispersions), topical formulations (gels, ointments, drops,creams), liposomal formulations (such as those described in: U.S. Pat.No. 9,629,804 B2; US 2012/0251618 A1; WO 2014/152211; US 2016/0038432A1).

In some embodiments, the in vitro synthesized RNA product can bedelivered into the cells by packaging them into nanoparticles such ascationic lipids and polymers, non-viral carriers like protamine. Directintroduction of the RNA into the cell using microinjection,electroporation, sonoporation can also be implemented. The delivery(localized or systemic) and the packaging of the RNA (with or withoutmodifications) can be performed at temperatures optimal for the deliveryapproach or the formulation used (such as those described in: U.S. Pat.No. 9,629,804 B2; US 2012/0251618 A1; WO 2014/152211; US 2016/0038432A1; US 2016/0032316 A1; U.S. Pat. No. 9,597,413 B2; US 2012/0258176).

A therapeutic formulation is also provided. In some embodiments, theformulation may comprise: (a) an RNA product produced by transcribing atemplate DNA using a thermostable RNA polymerase at a temperature ofgreater than 44° C.; and (b) pharmaceutically acceptable excipient.Consistent with the above, the formulation may be made in the absence ofa step that removes dsRNA from the RNA product.

Also provided is an RNA product produced by transcribing a template DNAusing a thermostable RNA polymerase at a temperature of greater than 44°C., for use as a medicament. Also provided is a therapeutic formulationof the invention, for use as a medicament.

Also provided is a method comprising: (a) transcribing a template DNAwith a thermostable RNA polymerase at a temperature of greater than 44°C. to produce an RNA product; and (b) measuring an immunostimulatoryeffect of the RNA product in the absence of a step that removes dsRNAfrom the RNA product. As discussed above, the immunostimulatory effectof an RNA product can be measured by introducing the RNA product tomammalian cells and measuring the expression of markers for innateimmunity (e.g., interferons and cytokines, among many others) by thecells. These cells may be in vitro, in vivo or ex vivo.

In some embodiments, the methods and compositions described herein maybe used to make polyribonucleotides which when transfected intoeukaryotic cells or prokaryotic cells in vivo or in vitro can change thecell phenotype by production of proteins or by affecting expression oftargets in the cell. This is best achieved if one can avoid generatingan immunostimulatory response (triggered by dsRNA) that would underminethe viability of the target cells.

RNA products of IVT can be used for encoding proteins such as antigensfor vaccines, for cancer immunotherapies (such as those described in:U.S. Pat. No. 8,217,016 B2; US 2012/0009221 A1; US 2013/0202645A1; U.S.Pat. No. 9,587,003 B2; Sahin, et al., (2014); allergy tolerance (such asthose described in Sahin, et al., (2014), for producing recombinant ornaturally occurring protein for protein replacement therapeutics (suchas those described in: US 2016/0032316 A1; US 2016/0032316; U.S. Pat.No. 8,680,069; WO 2013/151736; WO 2014/152940; U.S. Pat. Nos. 9,181,321;9,220,792 B2; 9,233,141 B2; Sahin, et al., (2014)), supplementationtherapeutics (such as those described in Sahin, et al., (2014)), cellreprogramming (such as those described in: US 2011/0143436 A1; U.S. Pat.Nos. 8,802,438; 9,371,544; WO/2009077134 A2; Sahin, et al., (2014)),genome editing/engineering (such as those described in Sahin, et al.,(2014)).

EMBODIMENTS

Embodiment 1. A method, comprising: (a) transcribing a template DNA witha thermostable RNA polymerase at a temperature of greater than 44° C. toproduce an RNA product; and (b) introducing the RNA product intomammalian cells, wherein the method is done in the absence of a stepthat removes any dsRNA from the RNA product between steps (a) and (b).

Embodiment 2. The method of embodiment 1, wherein the RNA productadministered in (b) is less immunostimulatory than a control RNA productproduced by transcribing the template DNA with the polymerase at atemperature of 37° C., wherein the control RNA is introduced into thecells in the absence of a step that removes any dsRNA from the controlRNA product.

Embodiment 3. The method of any prior embodiment, wherein thetranscribing is done at a temperature of at least 50° C.

Embodiment 4. The method of any prior embodiment, wherein thethermostable RNA polymerase is a variant of a bacteriophage RNApolymerase.

Embodiment 5. The method of any prior embodiment, wherein thethermostable RNA polymerase is a variant of the wild type T7 RNApolymerase of SEQ ID NO:1.

Embodiment 6. The method of any prior embodiment, including one or moreof the following: (i) the RNA product encodes a therapeutic protein,(ii) the RNA product does not include pseudouridine and/or (iii) the RNAproduct is preferably not delivered in a virus particle.

Embodiment 7. The method of any prior embodiment, wherein the RNAproduct is a therapeutic RNA.

Embodiment 8. The method of any prior embodiment, wherein the RNAproduct is selected from a guide RNA, a short hairpin RNA, a siRNA, amicroRNA, a long noncoding RNA, a mRNA encoding a recombinant protein ora native protein, an RNA containing modified nucleotides, and a cappedmRNA.

Embodiment 9. The method of any prior embodiment, further comprisingassaying the RNA product of step (a) for the presence of dsRNA, withoutperforming a step that removes any dsRNA from the RNA product.Embodiment 10. The method of any prior embodiment, wherein the cells arecells cultured in vitro.

Embodiment 11. The method of any of embodiments 1-9, wherein theintroducing is done by administering the RNA product to a subject invivo.

Embodiment 12. The method of any of embodiments 1-9, wherein the cellsare ex vivo.

Embodiment 13. A method, comprising: combining an RNA product made bytranscribing a template DNA with a thermostable RNA polymerase at atemperature of greater than 44° C. with a pharmaceutically acceptableexcipient to produce a formulation.

Embodiment 14. The method of embodiment 13, further comprisingadministering the formulation to a subject.

Embodiment 15. The method of any of embodiments 13 and 14, wherein theRNA product is combined with the pharmaceutically acceptable excipientin the absence of a step that removes any dsRNA from the RNA product.

Embodiment 16. A therapeutic formulation comprising: (a) an RNA productproduced by transcribing a template DNA using a thermostable RNApolymerase at a temperature of greater than 44° C.; and (b)pharmaceutically acceptable excipient.

Embodiment 17. A method, comprising: (a) transcribing a template DNAwith a thermostable RNA polymerase at a temperature of greater than 44°C. to produce an RNA product; (b) measuring the immunogenicity of theRNA product in the absence of a step that removes any dsRNA from the RNAproduct.

EXAMPLES

Aspects of the present teachings can be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

Synthesis of IVT mRNA—IVT reactions were performed in 41 mM Tris-HCl pH8.0, 50 mM NaCl, 19 mM MgCl₂, 5.5 mM DTT, 1 mM spermidine, 4 mM of eachribonucleotide, 4.15 units/5 mL yeast inorganic pyrophosphatase, 1000units/mL murine ribonuclease inhibitor, 30 nM DNA template and 30 nM T7RNA polymerase. The DNA template was plasmid DNA that was linearizedusing restriction endonucleases at specific sites downstream of the T7promoter. Reactions were performed at various temperatures ranging from37° C. to 55° C. For the synthesis of modified mRNA, UTP was replacedwith triphosphate derivatives of pseudouridine (Trilink Biotechnologies,San Diego, Calif.) in the IVT reaction. A 125-nt poly(A) tail wastemplate-encoded in mRNAs that were used for transfection experiments.IVT mRNAs were processed through a spin column (MEGAClear™, ThermoFisher Scientific, Waltham, Mass.) to remove unincorporated nucleotidesbefore performing capping reactions or HPLC purification. Capped mRNAswere generated for the transfection experiments using vaccinia cappingenzyme (New England Biolabs, Ipswich, Mass.), and 2′ O-methyltransferase(New England Biolabs, Ipswich, Mass.) was added to the reaction toattain the Cap1 structure required for efficient translation.

dsRNA immunoblot—IVT RNAs were blotted onto Nytran™ SuPerCharge (SPC)Blotting Membranes (GE Healthcare, Marlborough, Mass.). The driedmembranes were blocked for at least 1 hour in blocking buffer (TBSBlotto, Santa Cruz Biotechnologies, Dallas, Tex.) and then incubatedwith mAb-J2 (1:500 dilution; English and Scientific Consulting,Budapest, Hungary) for at least 4 hours. IRDye-800-conjugated donkeyanti-mouse secondary antibody (1:5000; LiCor, Lincoln, Nebr.) was usedfor detection using an Odyssey imaging system (LiCor, Lincoln, Nebr.).

HPLC purification of IVT RNA—HPLC purification of IVT RNA was performedas described previously (Kariko, et al., (2011); Weissman, et al.,(2012)). RNA from IVT reactions was loaded onto an analytical columnwith a matrix composed of alkylated non-porouspolystyrene-divinylbenzene copolymer microspheres that was obtained fromTransgenomic, Omaha, Nebr. The column was equilibrated with 38% buffer B(0.1 M triethylammonium acetate pH 7.0, 25% acetonitrile) and loadedwith the IVT RNA (10 μg per run) followed by a linear gradient of bufferB (38% to 65%). Fractions were collected for the major IVT RNA peak(II), and the fractions were desalted and concentrated using Amicon®Ultra-15 centrifugation units (EMD Millipore, Billerica, Mass.). RNA wasdiluted with nuclease-free water and subjected to capping.

Cell culture and transfection—hDCs were cultured in lymphocyte growthmedium with 50 ng/mL GM-CSF and 50 ng/mL IL-4 (Lonza, Portsmouth, N.H.)for four days prior to transfection. HEK293 cells were cultured inDulbecco's modified Eagle's medium (DMEM) supplemented with 2 mML-glutamine and 10% fetal calf serum (Thermo Fisher Scientific, Waltham,Mass.). Capped poly-adenylated IVT mRNA (500 ng) was complexed witheither Lipofectamine (Thermo Fisher Scientific, Waltham, Mass.) orTransIT®. Transfection was performed as recommended by the manufacturer.

Translation efficiency—The translation efficiency was measured asrelative luciferase activity from the transfected IVT Cluc NotI mRNA, asmeasured from the supernatant of transfected cells using the BioLux®Cypridina Luciferase Assay Kit (New England Biolabs, Ipswich, Mass.).Luminescence was measured using the Centro LB 960 Microplate Luminometerfrom Berthold Technologies (Wildbad, Germany). For each reaction, 20 μLof the supernatant was assayed 12 hours after transfection. The resultsin triplicate showed that the translation efficiency for the IVT RNAsynthesized by thermostable T7 RNA polymerase at 55° C. was similar tothe translation efficiency of IVT RNA synthesized by wild type T7 RNApolymerase at 37° C. and by IVT RNA synthesized by wild type T7 RNApolymerase at 37° C. after an HPLC column treatment at this incubationtime (where each sample provided 300,000-400,000 relative luciferaseunits). The negative controls (TransIT), PBS, Polyinosinic-polycytidylicacid (Polyl:C) synthetic dsRNA (Invivogen, San Diego, Calif.), andResiquimod (R848) small molecule immune activator (Invivogen, San Diego,Calif.)) all were consistently negative with zero detectable relativeluciferase units.

Immunogenicity assays—Immunogenicity was measured by ELISAs as describedpreviously (Kariko et al., (2011); Weissman, et al., (2012)).Supernatant from cells that were transfected with IVT mRNA was harvested24 hours after transfection and assayed for levels of IFN-α (PBLInterferon Source, Piscataway, N.J.) (for TransIT-complexed RNA) andTNF-α (Thermo Fisher Scientific, Waltham, Mass.) (forLipofectamine-complexed RNA). A standard curve using purified IFN-α orTNF-α was used to quantify the cytokines.

Results of experiments performed using the protocols described above areshown in FIGS. 1A-1B, 2A-2B, 3, 4 and 5A-5B. These results show that IVTat a temperature greater than 44° C. (e.g., at a temperature of greaterthan 44° C.) results in a product that has less dsRNA and is lessimmunogenic than a product transcribed at a lower temperature (e.g., atemperature of 37° C. or below).

What is claimed is: 1.-16. (canceled)
 17. A method for producing an RNAproduct with reduced immunogenicity, comprising: (a) combining a DNA anda thermostable RNA polymerase at a temperature of greater than 44° C.,the RNA polymerase variant having an amino acid sequence that has atleast 90% sequence identity with SEQ ID NO:1 and optionally having anamino acid substitution at a position corresponding to position 388 andat a position corresponding to position 567; and (b) producing the RNAproduct comprising a single strand RNA having reduced immunogenicitycompared with an RNA product produced by an RNA polymerase at 37° C. 18.A method according to claim 17, further comprising combining the singlestranded RNA with a pharmaceutically acceptable excipient.
 19. A methodaccording to claim 18, further comprising introducing the formulationinto a mammalian cell.
 20. The method of claim 18, further comprisingadministering the formulation to a subject.
 21. The method of claim 17,wherein the transcribing is done at a temperature of at least 50° C. 22.The method of claim 17, wherein the thermostable RNA polymerase is avariant of a bacteriophage RNA polymerase.
 23. The method of claim 17,wherein the thermostable RNA polymerase is a variant of the wild type T7RNA polymerase of SEQ ID NO:1.
 24. The method of claim 17, wherein theRNA product encodes a therapeutic protein.
 25. The method of claim 17,wherein the RNA product is a therapeutic RNA.
 26. The method of claim17, wherein the RNA product is selected from a guide RNA, a shorthairpin RNA, a siRNA, a microRNA, a long noncoding RNA, a mRNA encodinga recombinant protein or a native protein, an RNA containing modifiednucleotides, and a capped mRNA.